Recently, next-generation DNA sequencers have attracted attention as sequencing techniques for DNA nucleotide sequences (DNA sequencing) which have a higher parallel processing capability than DNA sequencers by Sanger method using capillary electrophoresis. Next-generation DNA sequencers achieve ultra-parallel processing by extending DNA fragments to be sequenced spotted highly densely on a substrate and detecting the luminescence of the extension reaction.
NPL 1 discloses a DNA sequencing technique of a next-generation DNA sequencer based on fluorescence detection. Reaction spots in which identical DNA fragments are clustered densely by amplification treatment are arranged highly densely on a glass sample substrate. When four kinds of base (A, T, G and C) labeled with four kinds of fluorophore are introduced to the substrate, a base complementary to that of the DNA fragments is incorporated by extension reaction by polymerase. Because the 3′-terminal of each fluorescently labeled base is modified with a functional group (terminator) for inhibiting the extension reaction, only one base is incorporated in one DNA fragment. After the extension reaction, excess free bases are washed out, and then the fluorescence emitted from each reaction spot is detected as a fluorescent spot and the kind of fluorophore is identified by the color. After the fluorescence detection, the terminator and the fluorophore are removed from each DNA fragment by chemical reaction so that the next base would be incorporated. By repeating the extension reaction, fluorescence detection and removal of the terminator one by one, about 100 bases of the sequence of the DNA fragments are determined.
NPL 2 discloses a DNA sequencing technique of a next-generation DNA sequencer based on detection of chemical luminescence (pyrosequencing). Beads which each have a diameter of about 30 μm wand carry identical DNA fragments fixed thereon by amplification treatment are contained in wells each having a diameter of about 50 μm. A closely packed honeycomb structure of reaction spots having such a well structure is on a sample substrate. The substrate faces an image sensor through optic fibers and is fixed in such a way that the light from a reaction spot is always detected by a same pixel of the image sensor. Although this structure makes sequencing easy, it is difficult to scan two or more fields and process the data in parallel. When a kind of base (for example A) is introduced to a DNA fragment on a bead, the base is incorporated by the extension reaction by polymerase when the complementary base is A. Because the beads are surrounded by a luciferase luminescent agent which emits light by pyrophosphoric acid, the extension can be recognized by detecting the luciferase luminescence emitted from the reaction spots while the extension reaction progresses by the image sensor. Theoretically, the luciferase luminescence amount is in proportion to the amount of pyrophosphoric acid, and thus a luciferase luminescence amount in proportion to the number of incorporated bases is detected in case of a homopolymer. By repeating the above extension reaction for A, T, G and C, around 400 bases of the sequence of the DNA fragments are determined. Because no terminator is used in the above method, it is necessary to introduce the four kinds of base separately to the substrate.